Rapid test for Lyme bacteria

ABSTRACT

The subject matter disclosed herein provides a method and a device for detection of one or more bacteria in a sample.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The ASCII copy, created on Feb. 7, 2019, isnamed 51557-002004_Sequence_Listing_01.13.21_ST25 and is bytes in size.

All patents, patent applications and publications cited herein arehereby incorporated by reference in their entirety. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

BACKGROUND OF THE INVENTION

Lyme disease, also known as Borreliosis is a debilitating conditioncaused by the bacterium Borrelia burgdorferi transmitted by the Deertick (also known as the Blacklegged Tick). The Deer tick may containamong other parasites, viruses and bacteria, a bacterium called Borreliaburgdorferi, which causes Lyme disease. Ticks can get infected withBorrelia burgdorferi from the white footed mouse, although ticks are notknown to get sick from the infections. Only female Deer ticks can passon Lyme disease due to the fact that only females feed on blood, whichcan allow for the bacterial infection to spread. The majority of tickbites that result in Borrelia burgdorferi infection and Lyme are causedby Nymphs, immature ticks that are less than 2 mm in size. Nymphs feedpredominantly during the spring and summer months which often coincideswith an increase in tick bites reported. A tick can attach itself to anypart of the human body, although they are most commonly found in warmareas such as behind the ear lobes or armpits. When a tick attaches ontoa human body it transmits the Borrelia burgdorferi bacterium into thebloodstream of the host. According to the Centers for Disease Control(CDC), a tick must be attached for 36 to 48 hours before it transmitsLyme disease.

Symptoms of Lyme disease include an infection related bulls-eye rashreferred to as erythema migrans, pains and swelling of the joints, aswell as fever and fatigue. An accurate and early detection of Lymedisease is critical to effective treatment. Erythema migrans, the mostdistinctive symptom and reliable way of clinically diagnosing Lymedisease, is only present early during the infection cycle and may notappear in all infected individuals. Other clinical symptoms that havebeen associated with Lyme disease, such as Bell's palsy, are notspecific enough to determine early clinical diagnosis in the absence oferythema migrans.

Antibiotic treatment for Lyme disease is very effective. Only about halfof Deer ticks are carriers of Borrelia. Treatment for Lyme diseaseshould be started as soon as possible after a tick bite, but unnecessarytreatment with antibiotics can cause significant problems such asinduction of resistance of common gut bacteria and allergic reactions.Current tests for identification of Borrelia-carrying ticks are verytime consuming.

Accordingly, there remains a need in the art for rapid and specificassays for detecting Borrelia burgdorferi antigens in ticks in order tobe able to treat only patients that have been bitten by a carrier tick.

SUMMARY OF THE INVENTION

In certain aspects, the invention provides a method for detecting one ormore vectors in a sample comprising homogenizing a sample on a samplepad, applying biotinylated analogue protein, streptavidin-gold, and flowbuffer to homogenized sample to obtain homogenized sample solution,allowing for capillary flow of said homogenized sample solution to occuralong an elongated nitrocellulose strip contacting at a first short edgesaid sample pad, allowing homogenized sample solution to contact a firstread-out line on the nitrocellulose strip and a second read-out line onthe nitrocellulose strip, wherein first read-out line comprises at leastone vector-specific antibody, further wherein second read-out linecomprises biotinylated albumin and collecting excess sample in cellulosewick contacting a second short edge of said nitrocellulose strip.

In certain aspects, the inventions provides a device for detecting oneor more vectors in a sample comprising a holder with a top partcomprising at least one sample opening and at least one observationopening and a continuous bottom part, a top glass slide and a bottomglass slide disposed between the top part and the bottom part of saidholder such that the holder encases the top glass slide and the bottomglass slide, at least one sample pad disposed on top of the bottom glassslide such that at least a portion of the at least one sample pad is notcovered by the top glass slide and at least a portion of the at leastone sample pad is in contact with a first short edge of at least oneelongated nitrocellulose strip and at least a portion of the sample padaligns with at least one sample opening on the top part of the holder,wherein a first portion of said at least one nitrocellulose strip ispre-loaded with at least one vector-specific antibody and a secondportion of the at least one nitrocellulose strip is pre-loaded withbiotinylated streptavidin, further wherein the at least onenitrocellulose strip is disposed between the top glass slide and thebottom glass slide and at least one cellulose wick disposed in contactwith a second short edge of the at least nitrocellulose strip.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows a diagram of a lateral flow immunoassay.

FIG. 2 shows a diagram of an assay for the detection of Borreliaburgdorferi.

FIG. 3 shows a positive test for the presence Borrelia burgdorferi.

FIG. 4 shows a negative test for the presence Borrelia burgdorferi.

FIG. 5 shows a diagram of a QuickLyme device for detection of Borreliaburgdorferi.

DETAILED DESCRIPTION OF THE INVENTION

In certain aspects, the invention provides a method for detecting one ormore Borrelia burgdorferi proteins in a sample comprising homogenizing asample on a sample pad, applying biotinylated analogue protein,streptavidin-gold, and flow buffer to homogenized sample to obtainhomogenized sample solution, allowing for capillary flow of saidhomogenized sample solution to occur along an elongated nitrocellulosestrip contacting at a first short edge said sample pad, allowinghomogenized sample solution to contact a first read-out line on thenitrocellulose strip and a second read-out line on the nitrocellulosestrip, wherein first read-out line comprises at least one Borreliaburgdorferi protein-specific antibody, further wherein second read-outline comprises biotinylated albumin and collecting excess sample incellulose wick contacting a second short edge of said nitrocellulosestrip.

In certain aspects, the inventions provides a device for detecting oneor more Borrelia burgdorferi proteins in a sample comprising a holderwith a top part comprising at least one sample opening and at least oneobservation opening and a continuous bottom part, a top glass slide anda bottom glass slide disposed between the top part and the bottom partof said holder such that the holder encases the top glass slide and thebottom glass slide, at least one sample pad disposed on top of thebottom glass slide such that at least a portion of the at least onesample pad is not covered by the top glass slide and at least a portionof the at least one sample pad is in contact with a first short edge ofat least one elongated nitrocellulose strip and at least a portion ofthe sample pad aligns with at least one sample opening on the top partof the holder, wherein a first portion of said at least onenitrocellulose strip is pre-loaded with at least one Borreliaburgdorferi protein-specific antibody and a second portion of the atleast one nitrocellulose strip is pre-loaded with biotinylatedstreptavidin, further wherein the at least one nitrocellulose strip isdisposed between the top glass slide and the bottom glass slide and atleast one cellulose wick disposed in contact with a second short edge ofthe at least nitrocellulose strip.

Lyme disease is an extremely harmful and painful disease that can beeasily treated with antibiotics. However, as with all medication therecan be undesired side-effects. Additionally, it is not recommended thatmedication be taken unless absolutely necessary. Therefore, whenexperiencing a tick bite, it is important to know if the tick carriesLyme disease, which can be transmitted to human subjects. The subjectmatter disclosed herein provides a method for rapid and robust detectionof Lyme disease-associated bacteria in ticks. This can inform a medicalprofessional as to possible course of treatment.

Lateral Flow Immunoassay

Lateral Flow Immunoassay is a simple and cost-effective methodology fordetecting a target protein in a liquid sample. Tests based on thismethodology can be used at a point of care center or for at-hometesting. The technology incorporates a series of capillary beds, whichhave the capacity to transport fluids spontaneously. FIG. 1 shows alateral flow immunoassay device positioned on a glass microscope slide(2.1). The device can include capillary beds consisting of glass fiber,nitrocellulose, or cellulose. Capillary beds take advantage of thecapillary action displayed by liquids, which is the ability of liquidsto flow through narrow spaces even in opposition to external forces.Capillary action can be observed in the drawing up of liquids intomaterials such as fibers or cellulose. It occurs due to intermolecularforces between the liquid and surrounding solid surfaces. If thediameter of the space is sufficiently small, then the combination ofsurface tension of the liquid and the adhesive forces between the liquidand the surrounding solid surface act to propel the liquid into thenarrow space. A glass fiber sample pad (2.2) can be the first element inthe capillary bed series and may hold any excess fluid from a sampleapplied on it. The sample can migrate in a unidirectional flow from thesample pad (2.2) along a nitrocellulose strip (2.3) as shown in FIG. 1 .In the course of capillary flow, the sample can come into contact withone or more read-out lines. FIG. 1 exemplifies two read-out lines, atest line (2.4), which can be pre-loaded with an antibody specificallyrecognizing an antigen of interest, and a control line (2.5), which canbe pre-loaded with biotinylated albumin with the capacity tospecifically bind streptavidin-gold. The control line (2.5) can changecolor when streptavidin-gold comes in contact with the pre-loadedbiotinylated albumin, indicating that the testing is proceeding underfavorable conditions for reliable results. The test line (2.4) canchange color only in the presence of Borrelia burgdorferi-specificbiotinylated peptide, indicating the absence of the bacteria in thesample. As shown in FIG. 1 test line (2.7) may not change colorindicating presence of Borrelia burgdorferi from sample, as Borreliacompetes with the biotinylated Borrelia peptide. After migrating alongthe nitrocellulose strip (2.3) and passing through the read-out lines(2.4; 2.4; 2.7), the sample enters the final porous material of theseries, the cellulose wick (2.6), which functions as a waste containeras shown in FIG. 1 .

There are two main types of lateral flow immunoassays. A “sandwichassay” and a “competitive assay”. In a “sandwich assay”, a migratingsample first encounters a conjugated antibody, which can be labelledwith an antibody specific to the target protein. If the target proteinis present in the sample, the conjugated antibody can bind to it andsubsequently reach the test line. The test line may also containantibodies specific to the target protein and will change color uponreaction with target protein. Thus, the target protein is “sandwiched”between two antibodies. In “competitive assays”, the target protein hasto compete with an added analogue in the sample in order to bindspecific antibodies on the test line. In competitive assays, anon-labelled target protein in a sample, such as a homogenized ticksample, competes with labelled proteins to bind an antibody. This typeof assay also uses a color change of the test line as a read-out.

The subject matter disclosed herein relates to detection of an antigensuch as the bacterium Borrelia burgdorferi in a sample using acompetitive lateral flow immunoassay. In some embodiments, the antigencan be a Borrelia burgdorferi protein or an analogue thereof. In someembodiments, the antigen can be the variable major protein (VMP)-likesequence (Vls) E lipoprotein (VlsE) protein, the antigenic variationprotein of Borrelia burgdorferi. In some embodiments, the antigen can bea C6 peptide. In some embodiments, the C6 peptide reproduces a sequenceof the invariable region 6 of the VlsE protein. In some embodiments, theC6 peptide is a synthetic peptide of a single 25 amino acid sequence ofthe Borrelia burgdorferi VlsE protein. The C6 peptide can be used with ahigh degree of sensitivity as a test antigen in a lateral flowimmunoassay. In some embodiments, the sample includes one or morehomogenized ticks. In some embodiments, the sample includes one or moretick-associated proteins.

QuickLyme Test

The subject matter disclosed herein relates to a test for detection ofLyme bacteria (Borrelia burgdorferi) in a sample (hereafter referred toas “QuickLyme” test). In some embodiments, the sample can be one or moreticks. In some embodiments, the sample can include isolated VlsE proteinor an analogue thereof. In some embodiments, the sample can include C6peptide. In some embodiments, the sample can include another shortsynthetic sequence peptide of Borrelia, which is biotinylated. In someembodiments, one or more ticks can be placed onto the sample pad in oneend of the QuickLyme nitrocellulose strip and homogenized. To aid incapillary flow and detection of tick proteins, about 3 to about 5 dropsof C6 peptide solution, about 3-5 drops of streptavidin-gold solution,and about 3 to about 5 drops of flow buffer can be added to thehomogenized sample or one or more ticks to obtain a homogenized samplesolution or tick-associated proteins. In some embodiments, the flowbuffer consists of Phosphate Buffered Saline, which can keep pH levelsstable in the sample during flow. Capillary forces pull the appliedsample and liquids across a nitrocellulose strip.

FIG. 2 shows a diagram of the protein components, which can beincorporated into a QuickLyme test for detecting the presence ofBorrelia burgdorferi in a sample. As shown in FIG. 2 , an antibodyspecific to the biotinylated C6 protein can be pre-loaded onto a test(sample) line of a nitrocellulose stip. In some embodiments, theantibody pre-loaded onto the test line can be specific for the VlsEprotein. In some embodiments, the antibody pre-loaded onto the test linecan be specific for another Borrelia burgdorferi protein or complex ofproteins. In some embodiments, the antibody pre-loaded onto the testline can be specific for one or more proteins associated with bacteriumother than Borrelia burgdorferi. As shown in FIG. 2 , the control linecan be pre-loaded with biotinylated albumin particles. When biotinylatedalbumin comes in contact with streptavidin-gold solution in the sample,the control line can become colored as a result of a binding reactionbetween streptavidin and biotin. In some embodiments, the control linecan become red in color. In the example shown in FIG. 2 , when VlsEprotein is present in the sample, it specifically binds to theanti-VlsE/C6 antibody in the test line and there is no color changeobserved because there is no color particle attached to the VlsEprotein, which is a proxy for Borrelia burgdorferi in the sample. Thus,when the QuickLyme test only presents with one colored line (the controlread-out line), it is indicative of presence of VlsE protein in thesample and possibly a Borrelia burgdorferi infection in tick sample asexemplified in FIG. 2 . The biotinylated C6 peptide added to the samplecan facilitate binding of streptavidin-gold to the anti-C6 antibody andindicate absence of VlsE protein, and thus absence of Borreliaburgdorferi, from the tick sample. In the example of FIG. 2 , when thetest and control read-out line become red, it is indicative of anegative test for VlsE protein and Borrelia burgdorferi infection. Theexample in FIG. 2 shows a “competitive assay” because if VlsE protein ispresent in the sample, it can compete with the added C6 peptide forbiding to the specific antibody.

In some embodiments, it is important to avoid drying up of sample bytimely adding reactants to sample pad.

Arthropods

Arthropods are invertebrates with segmented bodies and jointed limbs.Their exoskeleton or cuticles consist of chitin, a polymer ofglucosamine. Estimates of the number of arthropod species vary between1,170,000 and 5 to 10 million and account for over 80 percent of allknown living animal species. Medically, arthropods are more significantas carriers of diseases such as malaria, yellow fever, dengue fever, andelephantiasis (via mosquitos), African sleeping sickness (via tsetseflies), typhus fever (via lice), bubonic plague (via fleas), and RockyMountain spotted fever and Lyme disease (via ticks). Many diseases ofdomesticated animals are also transmitted by arthropods.

In one embodiment, the subject matter disclosed herein relates to amethod for detecting bacteria in a sample of one or more arthropods. Inone embodiment, the subject matter disclosed herein relates to a lateralflow immunoassay device for detecting an antigen in a sample of one ormore arthropods. In some embodiments, the sample includes a homogenizedarthropod. In some embodiments, the sample includes one or morehomogenized arthropods. In some embodiments, the sample includes two ormore homogenized arthropods from two or more species. In someembodiments, the arthropod is a mosquito. In some embodiments, thearthropod is a tick. In some embodiments, the arthropod is flea. In someembodiments, the arthropod carries a disease-causing antigen.

In some embodiments, the antigen causes malaria. In some embodiments,the antigen causes yellow fever. In some embodiments, the antigen causesdengue fever. In some embodiments, the antigen causes elephantiasis. Insome embodiments, the antigen causes African sleeping sickness. In someembodiments, the antigen causes typhus fever. In some embodiments, theantigen causes bubonic plague. In some embodiments, the antigen causesRocky Mountain spotted fever. In some embodiments, the antigen causesLyme disease.

Vectors and Vector-Bourne Diseases

Vectors are living organisms that can transmit infectious diseasesbetween humans or from animals to humans. Vector-borne diseases areinfections transmitted by the bite of infected arthropod species, suchas mosquitoes, ticks, triatomine bugs, sandflies, and blackflies.Arthropod vectors are cold-blooded (ectothermic) and thus especiallysensitive to climatic factors. However, climate is only one of manyfactors influencing vector distribution, such as habitat destruction,land use, pesticide application, and host density. Many vectors arebloodsucking insects, which ingest disease-producing microorganismsduring a blood meal from an infected host (human or animal) and laterinject it into a new host during their subsequent blood meal. Mosquitoesare the best known disease vector. Others include ticks, flies,sandflies, fleas, triatomine bugs and some freshwater aquatic snails.

Diseases that can be spread by mosquitoes include, but are not limitedto, chikungunya, dengue fever, lymphatic filariasis, rift valley fever,yellow fever, zika, malaria, lymphatic filariasis, Japaneseencephalitis, Lymphatic filariasis, and West Nile fever. Diseases thatcan be spread by sandflies include, but are not limited to,leishmaniasis, and sandfly fever (phelebotomus fever). Diseases that canbe spread by ticks include, but are not limited to, Crimean-Congohemorrhagic fever, Lyme disease, relapsing fever (borreliosis),rickettsial diseases (spotted fever and Q fever), tick-borneencephalitis, and tularaemia. Diseases that can be spread by triatominebugs include, but are not limited to, chagas disease (Americantrypanosomiasis). Diseases that can be spread by tsetse flies include,but are not limited to, Sleeping sickness (African trypanosomiasis).Diseases that can be spread by fleas include, but are not limited to,plague (transmitted by fleas from rats to humans), and rickettsiosis.Diseases that can be spread by black flies include, but are not limitedto, onchocerciasis (river blindness). Diseases that can be spread byaquatic snails include, but are not limited to, schistosomiasis(bilharziasis). Diseases that can be spread by lice include, but are notlimited to, typhus and louse-borne relapsing fever.

In one embodiment, the subject matter disclosed herein relates to amethod for detecting bacteria in a sample of one or more vectors. In oneembodiment, the subject matter disclosed herein relates to a lateralflow immunoassay device for detecting an antigen in a sample of one ormore vectors. In some embodiments, the sample includes a homogenizedvector. In some embodiments, the sample includes one or more homogenizedvectors. In some embodiments, the sample includes two or morehomogenized vectors from two or more species. In some embodiments, thevector is a mosquito. In some embodiments, the vector is a tick. In someembodiments, the vector is flea. In some embodiments, the vector carriesa disease-causing antigen.

In some embodiments, the antigen causes chikungunya. In someembodiments, the antigen causes dengue fever. In some embodiments, theantigen causes lymphatic filariasis. In some embodiments, the antigencauses rift valley fever. In some embodiments, the antigen causes yellowfever. In some embodiments, the antigen causes zika. In someembodiments, the antigen causes malaria. In some embodiments, theantigen causes lymphatic filariasis. In some embodiments, the antigencauses Japanese encephalitis. In some embodiments, the antigen causeslymphatic filariasis. In some embodiments, the antigen causes West Nilefever. In some embodiments, the antigen causes leishmaniasis. In someembodiments, the antigen causes Crimean-Congo hemorrhagic fever. In someembodiments, the antigen causes Lyme disease. In some embodiments, theantigen causes relapsing fever (borreliosis). In some embodiments, theantigen causes rickettsial diseases (spotted fever and Q fever). In someembodiments, the antigen causes tick-borne encephalitis. In someembodiments, the antigen causes tularaemia. In some embodiments, theantigen causes chagas disease (American trypanosomiasis). In someembodiments, the antigen causes sleeping sickness (Africantrypanosomiasis). In some embodiments, the antigen causes plague. Insome embodiments, the antigen causes rickettsiosis. In some embodiments,the antigen causes onchocerciasis (river blindness). In someembodiments, the antigen causes schistosomiasis (bilharziasis). In someembodiments, the antigen causes typhus. In some embodiments, the antigencauses louse-borne relapsing fever.

Antigens

In one embodiment, a homogenized sample can include one or more of thefollowing antigens:

-   -   Bacterium that causes Anaplasmosis    -   Parasite that causes Babesiosis    -   Bacterium that causes Borrelia mayonii    -   Bacterium that causes Borrelia miyamotoi    -   Bacterium that causes Bourbon virus    -   Virus that causes Colorado tick fever (CTF)    -   Bacterium that causes Ehrlichiosis    -   Virus that causes Heartland virus    -   Bacterium that causes Lyme disease    -   Virus that causes Powassan virus disease    -   Bacterium that causes Rickettsia parkeri rickettsiosis    -   Bacterium that causes Rocky Mountain spotted fever (RMSF)    -   Bacterium that causes STARI (Southern tick-associated rash        illness)    -   Bacterium that causes Tickborne relapsing fever (TBRE)    -   Bacterium that causes Tularemia    -   Bacterium that causes 364D ricketsiosis    -   Bacterium that causes Crimean-Congo hemorrhagic fever    -   Bacterium that causes Imported tickborne spotted fevers    -   Bacterium that causes Kyasanur forest disease    -   Most types of Borrelia    -   Bacterium that causes Omsk Hemorrhagic Fever (OHF)    -   Bacterium that causes Tickborne encephalitis (TBE)    -   Bacterium that causes African tick bite fever (ATBF)

In one embodiment, the subject matter disclosed herein is a method fordetecting the presence of one or more of the following antigens in asample:

-   -   Bacterium that causes Anaplasmosis    -   Parasite that causes Babesiosis    -   Bacterium that causes Borrelia mayonii    -   Bacterium that causes Borrelia miyamotoi    -   Bacterium that causes Bourbon virus    -   Virus that causes Colorado tick fever (CTF)    -   Bacterium that causes Ehrlichiosis    -   Virus that causes Heartland virus    -   Bacterium that causes Lyme disease    -   Virus that causes Powassan virus disease    -   Bacterium that causes Rickettsia parkeri rickettsiosis    -   Bacterium that causes Rocky Mountain spotted fever (RMSF)    -   Bacterium that causes STARI (Southern tick-associated rash        illness)    -   Bacterium that causes Tickborne relapsing fever (TBRE)    -   Bacterium that causes Tularemia    -   Bacterium that causes 364D ricketsiosis    -   Bacterium that causes Crimean-Congo hemorrhagic fever    -   Bacterium that causes Imported tickborne spotted fevers    -   Bacterium that causes Kyasanur forest disease    -   Most types of Borrelia    -   Bacterium that causes Omsk Hemorrhagic Fever (OHF)    -   Bacterium that causes Tickborne encephalitis (TBE)    -   Bacterium that causes African tick bite fever (ATBF)

In one embodiment, the subject matter disclosed herein is a device fordetecting the presence one or more of the following antigens in asample:

-   -   Bacterium that causes Anaplasmosis    -   Parasite that causes Babesiosis    -   Bacterium that causes Borrelia mayonii    -   Bacterium that causes Borrelia miyamotoi    -   Bacterium that causes Bourbon virus    -   Virus that causes Colorado tick fever (CTF)    -   Bacterium that causes Ehrlichiosis    -   Virus that causes Heartland virus    -   Bacterium that causes Lyme disease    -   Virus that causes Powassan virus disease    -   Bacterium that causes Rickettsia parkeri rickettsiosis    -   Bacterium that causes Rocky Mountain spotted fever (RMSF)    -   Bacterium that causes STARI (Southern tick-associated rash        illness)    -   Bacterium that causes Tickborne relapsing fever (TBRE)    -   Bacterium that causes Tularemia    -   Bacterium that causes 364D ricketsiosis    -   Bacterium that causes Crimean-Congo hemorrhagic fever    -   Bacterium that causes Imported tickborne spotted fevers    -   Bacterium that causes Kyasanur forest disease    -   Most types of Borrelia    -   Bacterium that causes Omsk Hemorrhagic Fever (OHF)    -   Bacterium that causes Tickborne encephalitis (TBE)    -   Bacterium that causes African tick bite fever (ATBF)

EXAMPLES Example 1

In the U.S., the New England region as well as areas of the PacificCoast are known to house large numbers of Deer ticks, also known as theblacklegged tick. Lyme disease can be extremely painful and althoughrare, the Centers for Disease Control and Prevention (CDC) has had 36reported deaths from Lyme disease. Because of this, it is extremelyimportant that there is a rapid method of early detection of tickscarrying the Borrelia bacteria. The State of Connecticut offers freetick tests, however, results can take two weeks or more.

The QuickLyme test is a faster, cheaper, and reliable way to test fordetecting bacterial antigens in ticks that only takes a few minutes andthere is no need to send specimens to a laboratory or be supervised by amedical professional. The test can consist of a nitrocellulose stripwith a sample pad on one side, and a wick on the opposite end of thenitrocellulose strip. In one embodiment, the sample is a syntheticprotein with the same structure as that of a Borrelia protein such asthe VlsE protein. VlsE can be applied to the sample pad with or withoutbiotinylated C6 protein. About 3-5 drops of streptavidin-gold and flowbuffer are added, and capillary forces pull the liquid to over testlines and towards the wick at the end. The lines give either a positiveor negative result, depending whether Borrelia (or the syntheticprotein) is present. In some embodiments, the QuickLyme test hasresearch applications, including surveying of the number of infectedticks in a certain area.

In one embodiment, the test procedure can proceed following the stepsoutlined below:

-   -   1. Gather materials and safety equipment including nitrile        gloves    -   2. Cut Nitrocellulose into 0.5 cm strips from 10 cm×10 cm sheets        and lay on microscope slides    -   3. Cut pads of glass fiber and cellulose into small rectangles    -   4. Dilute solutions    -   5. Carefully apply 3 μl of antibody and 3 μl of biotin albumin        in two separate lines using pipette across cellulose strip, then        let air dry        -   For a negative test (no Borrelia protein/VlsE), apply 20 μl            total of flow buffer containing 3 μl of C-6 peptide and 3 μl            of coloring solution (streptavidin-gold)        -   For a positive test (mimicking Borrelia bacteria presence),            apply 3 μl of VlsE protein in addition to 3 μl of C6 peptide            and 3 μl of coloring solution (streptavidin-gold)    -   6. Apply 500 μl of flow buffer to sample pad area after applying        reagents for both positive and negative test strips.

In one embodiment, the nitrocellulose strip is pre-wet with flow bufferto facilitate moving of the sample in the direction of flow.

As shown in FIG. 3 , a positive QuickLyme test for VlsE protein resultsin visualization of one red read-out line, which is the control line. Asshown in FIG. 4 , a negative test QuickLyme for VlsE results invisualization of two red read-out lines.

Materials and Methods

-   -   VlsE protein (VlsE Protein—Rockland Immunochemicals, Inc.,        Limerick, PA)    -   VlsE—Peptide-biotin        (Met-Lys-Lys-Asp-Asp-Gln-Ile-Ala-Ala-Ala-Ile-Ala-Leu-Arg-Gly-Met-Ala-Lys-Asp-Gly-Lys-Phe-Ala-Val-Lys-biotinamidocaproyl        (SEQ ID NO: 1)—GenScript, Piscataway, NJ)    -   VlsE antibody (rabbit anti-VlsE—Rockland Immunochemicals, Inc.,        Limerick, PA)    -   Biotin-Albumin (bovine albumin,        biotinamidocaproyl-labelled—Sigma Aldrich, Atlanta, GA)    -   Streptavidin-Gold (from Streptomyces avidinii, 20 nm particle        size—Sigma Aldrich, Atlanta, GA)    -   Phosphate buffered saline (PBS) pH 7.4 (sodium phosphate/NaPO4        50 mM, sodium chloride/NaCl 100 mM), (SigmaAldrich, Atlanta, GA)    -   Tween-20 (Polyoxyethylene-20-sorbitane        monolaurate—FisherScientific, Pittsburgh, PA)    -   Nitrocellulose blotting membrane (Amersham Protran Premium 0.45        um NC—GE Healthcare Life Sciences/Amersham, Pittsburgh, PA)    -   Cellulose Fiber Pads (EMD Millipore Corporation, Burlington, MA)    -   Glass Fiber Conjugate Pads (EMD Millipore Corporation,        Burlington, MA)    -   Microscope Glass Slides 1″×3″×1 mm (FisherScientific,        Pittsburgh, PA)    -   Single Channel Pipets, 10 ul, 20 ul, 200 ul, 1000 ul (Rainin,        Woburn, MA)    -   Polyethylene pipet tips, single use, 10 ul, 20 ul, 2000 ul, 1000        ul (Rainin, Woburn, MA)    -   Microcentrifuge tubes, 1.5 ml (Sigma Aldrich, Atlanta, GA)    -   Sterile deionized water (Sigma Aldrich, Atlanta, GA)    -   3D printer (Makerbot Replicator 2, Makerbot Industries,        Brooklyn, NY)    -   PLA-Filament, 1.75 mm (Polylactic acid resin, Makerbot        Industries, Brooklyn, NY)    -   Sketchup Software (Trimble Inc., Sunnyvale, CA)    -   MacBook Pro/OS X 10.12 (Apple Inc., Cupertino, CA)

Example 2

The subject matter disclosed herein relates to a QuickLyme device forLyme disease testing as shown in FIG. 5 . In one embodiment, theQuickLyme device can be manufactured following the steps outlinedherein. Using Sketchup software package on a Macbook Pro under OS X12.12, a holder (1.1) for the QuickLyme device is designed. On aMakerbot Replicator 2, both the top and bottom part of the holder (1.1)are printed using Makerbot PLA filament with the PLA standard printingtemperature and a layer resolution of 100 microns. In anotherembodiment, any three-dimensional design software and printer known inthe art can be used to manufacture a QuickLyme device. Top (1.5) andbottom (1.8) clear standard microscope glass slides are dip-cleaned in90% isopropyl alcohol, air dried and positioned between the top andbottom part of the holder (1.1). Using a razor blade, strips ofnitrocellulose (10 mm×80 mm) are cut and at least one piece ofnitrocellulose strip (1.7) is disposed between the top (1.5) and bottom(1.8) glass slides. Using a razor blade, glass fiber sample/reagent pads(10 mm×10 mm) are cut and at least one piece of sample pad (1.6) isdisposed on top of the bottom glass slide (1.8) such that it is notcovered by the top glass slide (1.5). Cellulose fiber wicks are cut to1.5×2.5 cm size and at least one cellulose wick (1.9) is placed underthe nitrocellulose strip (1.7) such that it is not covered by the bottomclass slide (1.8).

Onto the nitrocellulose strip (1.7), 5 μl of a 1:100 dilution of VlsEantibody in PBS are applied in a vertical line at 4 cm from the origin;at 5 cm from a distal end of the sample pad (1.6), 5 μl ofbiotin-albumin (1 mg/ml in PBS) are applied in a vertical line as well.The nitrocellulose strip is air dried for 30 minutes. Onto one edge ofthe glass fiber sample pad (1.6), 5 μl of biotinylated C6 peptidesolution in PBS are applied and air dried as well. In one embodiment,the holder (1.1) can be assembled as shown in FIG. 5 . A cleanmicroscope slide (1.8) is placed in the bottom part of the 3D-printedholder (1.1), a nitrocellulose strip (1.7) with VlsE antibody andbiotin-albumin is laid on top as indicated in FIG. 5 . A glass fibersample pad (1.6) containing peptide is placed at one end and a cellulosefiber wick (1.9) is placed on the opposite end of the nitrocellulosestrip (1.7). The strip assembly is covered with a clean microscope slideas indicated (1.5), overlapping the cellulose wick (1.9) and leaving thesample pad (1.6) accessible. The top part of the 3D-printed holder isplaced ensuring that a sample opening (1.2) is directly over the glassfiber sample pad (1.6) and the nitrocellulose strip (1.7) visiblethrough an observation opening (1.4) as shown in FIG. 5 .

During testing, sample can be applied onto the sample opening (1.2).When a tick is placed in the sample opening (1.2), the pestle (1.3) isused to manually homogenize the tick onto the sample pad before applyingflow buffer. In one embodiment, the sample may contain more than onetick. About 0.5 ml of phosphate-buffered saline with about 0.5% tween,pH 7.4 are added followed by about 0.75 ml of streptavidin-goldconjugate in the same buffer. In one embodiment, the sample buffer canbe applied on the one or more homogenized ticks to facilitate movementof tick-associated proteins through the nitrocellulose strip viacapillary action.

Through the observation opening, the nitrocellulose strip (1.7) can bemonitored for color development of the test and control lines which canoccur within 2-3 minutes. A strip showing two red lines indicates theabsence of Borrelia proteins from a sample. A strip showing only one redline indicates the presence or Borrelia proteins in a sample.

Materials

-   -   Makerbot 2 3D Printer+software    -   Glass fiber Pads    -   Cellulose Pads    -   Borrelia (VlsE-synthetic/non-harmful)    -   Borrelia antibody (anti-VlsE)    -   C6-Peptide    -   Coloring Solution (SAV-Gold)    -   Microscope Slides    -   Pipettes    -   Flow Buffer (Phosphate Buffered Saline can keep pH stable)

REFERENCES

-   1. “Lyme Disease.” Centers for Disease Control and Prevention, 19    Jan. 2018,-   2. Wong, Raphael C., and Harley Y. Tse. Lateral flow immunoassay.    Springer, 2010.-   3.    https://www.cdc.gov/ticks/tickbornediseases/TickborneDiseases-P.pdf-   4.    https://www.who.int/news-room/fact-sheets/detail/vector-borne-diseases

The invention claimed is:
 1. A device for detecting Borrelia sp. in asample comprising: a holder with a top part comprising at least onesample opening and at least one observation opening and a continuousbottom part; a top glass slide and a bottom glass slide disposed betweenthe top part and the bottom part of said holder such that the holderencases the top glass slide and the bottom glass slide; at least onesample pad disposed on top of the bottom glass slide such that at leasta portion of the at least one sample pad is not covered by the top glassslide and at least a portion of the at least one sample pad is incontact with a first short edge of at least one elongated nitrocellulosestrip and at least a portion of the sample pad aligns with at least onesample opening on the top part of the holder; wherein the at least onesample opening is shaped to receive a pestle for homogenization of thesample on the sample pad and is configured to constrain movement of thesample on the sample pad during homogenization of the sample followingapplication thereof to the sample pad; wherein a first portion of saidat least one nitrocellulose strip is pre-loaded with at least onevariable major protein (VMP)-like sequence (Vls) E lipoprotein (VlsE) C6peptide-specific antibody and a second portion of the at least onenitrocellulose strip is pre-loaded with biotinylated streptavidin;further wherein the at least one nitrocellulose strip is disposedbetween the top glass slide and the bottom glass slide; and at least onecellulose wick disposed in contact with a second short edge of the atleast one nitrocellulose strip.
 2. The device of claim 1, wherein thedevice is configured to detect Borrelia burgdorferi in the samplefollowing application of the sample to the sample pad.
 3. The device ofclaim 1, wherein the first portion of the nitrocellulose strip comprisesa first read out-line comprising the at least one C6 peptide-specificantibody.
 4. The device of claim 3, wherein the device is configured sothat the read-out line does not change color if Borrelia sp. is detectedin the sample.
 5. The device of claim 3, wherein the device isconfigured so that the read-out line changes color if Borrelia sp. isdetected in the sample.
 6. The device of claim 1, wherein the samplecomprises a tick or a portion thereof.